High affinity TGFβ nucleic acid ligands

ABSTRACT

Methods are described for the identification and preparation of high-affinity nucleic acid ligands to TGF beta . Included in the invention are specific RNA and ssDNA ligands to TGF beta 1 identified by the SELEX method. Also included are RNA ligands that inhibit the interaction of TGF beta 1 with its receptor.

RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. patent applicationSer. No. 07/714,131, filed Jun. 10, 1991, entitled Nucleic Acid Ligands,now U.S. Pat. No. 5,475,096, U.S. patent application Ser. No.07/931,473, filed Aug. 17, 1992, entitled Nucleic Acid Ligands, nowissued as U.S. Pat. No. 5,270,163, U.S. patent application Ser. No.07/964,624, filed Oct. 21, 1992, entitled Methods of Producing NucleicAcid Ligands, now U.S. Pat. No. 5,496,938, and U.S. patent applicationSer. No. 08/117,991, filed Sept. 8, 1993, abandoned, entitledHigh-Affinity Nucleic Acid Ligands Containing Modified Nucleotides. U.S.patent application Ser. No. 07/714,131 (now U.S. Pat. No. 5,475,096) isa Continuation-in-Part of U.S. patent application Ser. No. 07/536,428,filed Jun. 11, 1990, entitled Systematic Evolution of Ligands byEXponential Enrichment, now abandoned.

FIELD OF THE INVENTION

Described herein are methods for identifying and preparing high-affinitynucleic acid ligands to TGFβ. The method utilized heroin for identifyingsuch nucleic acid ligands is called SELEX, an acronym for SystematicEvolution of Ligands by EXponential enrichment. This invention includeshigh affinity nucleic acid ligands of TGFβ. Further disclosed are RNAand DNA ligands to TGFβ1. Also included are oligonucleotides containingnucleotide derivatives chemically modified at the 2'- positions ofpyrimidines. Additionally disclosed are RNA ligands to TGFβ1 containing2'-NH₂ -modifications or 2'-F-modifications. This invention alsoincludes high affinity nucleic acid inhibitors of TGFβ1. Theoligonucleotides of the present invention are useful as pharmaceuticalsor diagnostic agents.

BACKGROUND OF THE INVENTION

The transforming growth factor -β (TGFβ) polypeptides influence growth,differentiation, and gene expression in many cell types. The firstpolypeptide of this family that was characterized, TGFβ1 has twoidentical 112 amine acid subunits which are covalently linked. TGFβ1 isa highly conserved protein with only a single amine acid differencedistinguishing humans from mice. There are two other members of the TGFβgene family that are expressed in mammals. TGFβ2 is 71% homologous toTGFβ1(de Martin et al. (1987) EMBO J. 6:3673-3677), whereas TGFβ3 is 80%homologous to TGFβ1(Derynck et al. (1988) EMBO J 7:3737-3743). Thestructural characteristics of TGFβ1 as determined by nuclear magneticresonance (Archer et al. (1993) Biochemistry 32:1164-1171) agree withthe crystal structure of TGFβ2(Daopinet al. (1992) Science 257:369-374;Schlunegger and Grutter (1992) Nature 358:430-434).

Even though the TGFβ's have similar three dimensional structures, theyare by no means physiologically equivalent. There are at least threedifferent extracellular receptors, type I, II and III, involved intransmembrane signaling of TGFβ to cells carrying the receptors. Forreviews, see Derynck (1994) TIBS 19:548-553 and Massague (1990) Annu.Rev. Cell Biol 6:597-641). In order for TGFβ2 to effectively interactwith the type II TGFβ receptor, the type III receptor must also bepresent (Derynck (1994) TIBS 19:548-553). Vascular endothelial cellslack the type III receptor. Instead endothelial cells express astructurally related protein called endoglin (Cheifetz, et al. (1992) J.Biol. Chem. 267:19027-19030), which only binds TGFβ1 and TGFβ3 with highaffinity. Thus, the relative potency of the TGFβ's reflect the type ofreceptor expressed in a cell and organ system.

In addition to the regulation of the components in the multifactorialsignaling pathway, the distribution of the synthesis of TGFβpolypeptides also affects physiological function. The distribution ofTGFβ2 and TGFβ3 is more limited (Derynck et al. (1988) EMBO J7:3737-3743) than TGFβ1, eg. TGFβ3 is limited to tissues of mesenchymalorigin, whereas TGFβ1 is present in both mesenchymal and epithelialcells.

TGFβ1 is a multifunctional cytokine critical for tissue repair. Highconcentrations of TGFβ1 are delivered to the site of injury by plateletgranules (Assoian and Sporn, (1986) J Cell Biol. 102:1217-1223). TGFβ1initiates a series of events that promote healing including chemotaxisof cells such as leukocytes, monocytes and fibroblasts, and regulationof growth factors and cytokines involved in angiogenesis, cell divisionassociated with tissue repair and inflammatory responses. TGFβ1 alsostimulates the synthesis of extracellular matrix components (Roberts etal., (1986) Proc. Natl. Acad Sci USA 83:4167-4171; Sporn et al., (1983)Science 219:1329-1330; Massague, (1987) Cell 49:437-438) and mostimportantly for understanding the pathophysiology of TGFβ1, TGFβ1autoregulates its own synthesis (Kim et al., (1989) J Biol Chem264:7041-7045).

A number of diseases have been associated with TGFβ1 overproduction.Fibrotic diseases associated with TGFβ1 overproduction can be dividedinto chronic conditions such as fibrosis of kidney, lung and liver andmore acute conditions such as dermal scarring and restenosis. Synthesisand secretion of TGFβ1 by tumor cells can also lead to immunesuppression such as seen in patients with aggressive brain or breasttumors (Arteaga et al., (1993) J Clin Invest 92:2569-2576). The courseof Leishmanial infection in mice is drastically altered by TGFβ1(Barral-Netto, et al. (1992) Science 257:545-547). TGFβ1 exacerbated thedisease, whereas TGFβ1 antibodies halted the progression of the diseasein genetically susceptible mice. Genetically resistant mice becamesusceptible to Leshmanial infection upon administration of TGFβ1.

The profound effects of TGFβ1 on extracellular matrix deposition havebeen reviewed (Rocco and Ziyadeh, (1991) in Contemporary Issues inNephrology v23, Hormones, autocoids and the kidney. ed. Jay Stein,Churchill Livingston, NY pp391-410; Roberts et al., (1988) Rec. Prog.Hormone Res. 44:157-197) and include the stimulation of the synthesisand the inhibition of degradation of extracellular matrix components.Since the structure and filtration properties of the glomerulus arelargely determined by the extracellular matrix composition of themesangium and glomerular membrane, it is not surprising that TGFβ1 hasprofound effects on the kidney. The accumulation of mesangial matrix inproliferative glomerulonephritus (Border et al., (1990) Kidney Int.37:689-695) and diabetic nephropathy (Mauer et al., (1984) J. ClinInvest.74:1143-1155) are clear and dominant pathological features of thediseases. TGFβ1 levels are elevated in human diabetic glomerulosclerosis(advanced neuropathy) (Yamamoto et al., (1993) Proc. Natl. Acad. Sci.90:1814-1818). TGFβ1 is an important mediator in the genesis of renalfibrosis in a number of animal models (Phan (et al.), (1990) Kidney Int.37:426; Okuda et al., (1990) J. Clin Invest. 86:453). Suppression ofexperimentally induced glomerulonephritus in rats has been demonstratedby antiserum against TGFβ1 (Border et al., (1990) Nature 346:371) and byan extracellular matrix protein, decorin, which can bind TGFβ1 (Borderet al., (1992) Nature 360:361-363).

Too much TGFβ1 leads to dermal scar-tissue formation. Neutralizing TGFβ1antibodies injected into the margins of healing wounds in rats have beenshown to inhibit scarring without interfering with the rate of woundhealing or the tensile strength of the wound (Shah et al., (1992) Lancet339:213-214). At the same time there was reduced angiogenesis, reducednumber of macrophages and monocytes in the wound, and a reduced amountof disorganized collagen fiber deposition in the scar tissue.

TGFβ1 may be a factor in the progressive thickening of the arterial wallwhich results from the proliferation of smooth muscle cells anddeposition of extracellular matrix in the artery after balloonangioplasty. The diameter of the restenosed artery may be reduced 90% bythis thickening, and since most of the reduction in diameter is due toextracellular matrix rather than smooth muscle cell bodies, it may bepossible to open these vessels to 50% simply by reducing extensiveextracellular matrix deposition. In uninjured pig arteries transfectedin vivo with a TGFβ1 gene, TGFβ1 gene expression was associated withboth extracellular matrix synthesis and hyperplasia (Nabel et al.,(1993) Proc. Natl. Acad. Sci USA 90:10759-10763). The TGFβ1 inducedhyperplasia was not as extensive as that induced with PDGF-BB, but theextracellular matrix was more extensive with TGFβ1 transfectants. Noextracellular matrix deposition was associated with FGF-1 (a secretedform of FGF) induced hyperplasia in this gene transfer pig model (Nabel(1993) Nature 362:844-846).

There are several types of cancer where TGFβ1 produced by the tumor maybe deleterious. MATLyLu rat cancer cells (Steiner and Barrack, (1992)Mol. Endocrinol. 6:15-25) and MCF-7 human breast cancer cells (Arteagaet al (1993) Cell Growth and Differ. 4:193-201) became more tumorigenicand metastatic after transfection with a vector expressing the mouseTGFβ1. In breast cancer, poor prognosis is associated with elevated TGFβ(Dickson et al., (1987) Proc. Natl. Aced Sci. USA 84:837-841; Kasid etal., (1987) Cancer Res. 47:5733-5738; Daly et al., (1990) J Cell Biochem43:199-211; Barrett-Lee et al., (1990) Br. J Cancer 61:612-617; King etal., (1989) J Steroid Biochem 34:133-138; Welch et al., (1990) Proc.Nat. Acad Sci. 87:7678-7682; Walker et al (1992) Eur J Cancer238:641-644) and induction of TGFβ1 by tamoxifen treatment (Butta et al,(1992) Cancer Res 52:4261-4264) has been associated with failure oftamoxifen treatment for breast cancer (Thompson et al, (1991) Br. JCancer 63:609-614). Anti TGFβ1 antibodies inhibit the growth of MDA-23 1human breast cancer cells in athymic mice (Arteaga et al., (1993) J ClinInvest 92:2569-2576), a treatment which is correlated with an increasein spleen natural killer cell activity. CHO cells transfected withlatent TGFβ1 also showed decreased NK activity and increased tumorgrowth in nude mice (Wallick et al., (1990) J Exp Med 172:1777-1784).Thus, TGFβ1 secreted by breast tumors may cause an endocrine immunesuppression.

High plasma concentrations of TGFβ1 have been shown to indicate poorprognosis for advanced breast cancer patients (Anscher, et al. (1993) NEngl J Med 328:1592-8). Patients with high circulating TGFβ before highdose chemotherapy and autologous bone marrow transplantation are at nighrisk for hepatic veno-occlusive disease (15-50% of all patients with amortality rate up to 50%) and idiopathic interstitial pneumonitis(40-60% of all patients). The implication of these findings is 1) thatelevated plasma levels of TGFβ1 can be used to identify at risk patientsand 2) that reduction of TGFβ1 could decrease the morbidity andmortality of these common treatments for breast cancer patients.

A method for the in vitro evolution of nucleic acid molecules with highaffinity binding to target molecules has been developed. This method,Systematic Evolution of Ligands by EXponential enrichment, termed SELEX,is described in U.S. patent application Ser. No. 07/536,428, entitledSystematic Evolution of Ligands by Exponential Enrichment, nowabandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10,1991, entitled Nucleic Acid Ligands, now U.S. Pat. No. 5,475,096, U.S.patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitledNucleic Acid Ligands, now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated byreference. Each of these applications, collectively referred to hereinas the SELEX Patent Applications, describe a fundamentally novel methodfor making a nucleic acid ligand to any desired target molecule.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection theme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of nucleic acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound nucleic acids from those nucleic acidswhich have bound to target molecules, dissociating the nucleicacid-target complexes, amplifying the nucleic acids dissociated from thenucleic acid-target complexes to yield a ligand-enriched mixture ofnucleic acids, then reiterating the steps of binding, partitioning,dissociating and amplifying through as many cycles as desired to yieldhigh affinity nucleic acid ligands to the target molecule.

The basic SELEX method may be modified to achieve specific objectives.For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14,1992, entitled Method for Selecting Nucleic Acids on the Basis ofStructure, describes the use of SELEX in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled Photoselection ofNucleic Acid Ligands describes a SELEX based method for selectingnucleic acid ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993,entitled High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine, describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, termed "counter-SELEX." U.S. patent application Ser.No. 08/143,564, filed Oct. 25, 1993, entitled Systematic Evolution ofLigands by EXponential Enrichment: Solution SELEX, describes aSELEX-based method which achieves highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability ordelivery. Examples of such modifications include chemical substitutionsat the ribose and/or phosphate and/or base positions. SpecificSELEX-identified nucleic acid ligands containing modified nucleotidesare described in U.S. patent application Ser. No. 08/117,991, filed Sep.8, 1993, entitled High Affinity Nucleic Acid Ligands Containing ModifiedNucleotides, that describes oligonucleotides containing nucleotidederivatives chemically modified at the 5- and 2'-modifications ofpyrimidines, as well as specific RNA ligands to thrombin containing2'-amino modifications. U.S. patent application Ser. No. 08/134,028,supra, describes highly specific nucleic acid ligands containing one ormore nucleotides modified with 2'-amino (2'-NH₂), 2'-fluoro (2'-F),and/or 2'-O-methyl (2'-OMe). Each of these applications is specificallyincorporated herein by reference.

BRIEF SUMMARY OF THE INVENTION

The present invention includes methods of identifying and producingnucleic acid ligands to transforming growth factor beta (TGFβ) and thenucleic acid ligands so identified and produced. For the purpose of thisapplication, TGFβ includes human TGFβ1, TGFβ2, and TGFβ3 and TGFβ's thatare substantially homologous thereto. By substantially homologous it ismeant a degree of amino acid sequence identity of 70% or more. Inparticular, RNA sequences are provided that are capable of bindingspecifically to TGFβ1. Specifically included in the invention are theRNA ligand sequences shown in Table 3 (SEQ ID NOS:12-42). These RNAligand sequences include both pre and post SELEX modifications as shownin Table 2. Also included in this invention are RNA ligands of TGFβ1that inhibit the function of TGFβ1.

Also included in the invention are ssDNA ligands to TGFβ1. Specificallyincluded in the invention are the ssDNA ligand sequences shown in Table6 (SEQ ID NOS:55-89).

Further included in this invention is a method of identifying nucleicacid ligands and nucleic acid ligand sequences to TGFβ comprising thesteps of (a) preparing a candidate mixture of nucleic acids, (b)contacting the candidate mixture of nucleic acids with TGFβ, (c)partitioning between members of said candidate mixture on the basis ofaffinity to TGFβ, and (d) amplifying the selected molecules to yield amixture of nucleic acids enriched for nucleic acid sequences with arelatively higher affinity for binding to TGFβ.

More specifically, the present invention includes the RNA and ssDNAligands to TGFβ identified according to the above-described method,including those ligands shown in Tables 3 and 6 (SEQ ID NOS: 12-42;55-89). Also included are nucleic acid ligands to TGFβ that aresubstantially homologous to any of the given ligands and that havesubstantially the same ability to bind TGFβ and inhibit the function ofTGFβ. Further included in this invention are nucleic acid ligands toTGFβ that have substantially the same structural form as the ligandspresented herein and that have substantially the same ability to bindTGFβ and inhibit the function of TGFβ.

The present invention also includes other modified nucleotide sequencesbased on the nucleic acid ligands identified herein and mixtures of thesame.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the binding analysis of the 40D7 DNA library for TGFβ1.Binding data obtained from Round 19 (triangles) and Round 0 (circles)are shown.

FIG. 2 shows the results of the PAI-luciferase assay of TGFβ1 (10 pM)incubated with oligonucleotides (0.1 μM) or anti-TGFβ (60 μg/ml).

DETAILED DESCRIPTION OF THE INVENTION

This application describes high-affinity nucleic acid ligands to TGFβidentified through the method known as SELEX. SELEX is described in U.S.patent application Ser. No. 07/536,428, entitled Systematic Evolution ofLigands by EXponential Enrichment, now abandoned, U.S. patentapplication Ser. No. 07/714,131, filed Jun. 10, 1991, entitled NucleicAcid Ligands, now U.S. Pat. No. 5,475,096, U.S. patent application Ser.No. 07/931,473, filed Aug. 17, 1992, entitled Nucleic Acid Ligands, nowU.S. Pat. No. 5,270,163, (see also WO 91/19813). These applications,each specifically incorporated herein by reference, are collectivelycalled the SELEX Patent Applications.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning. 4) Those nucleic acids selected duringpartitioning as having the relatively higher affinity to the target arethen amplified to create a new candidate mixture that is enriched innucleic acids having a relatively higher affinity for the target. 5) Byrepeating the partitioning and amplifying steps above, the newly formedcandidate mixture contains fewer and fewer weakly binding sequences, andthe average degree of affinity of the nucleic acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield acandidate mixture containing one or a small number of unique nucleicacids representing those nucleic acids from the original candidatemixture having the highest affinity to the target molecule.

The SELEX Patent Applications describe and elaborate on this process ingreat detail. Included are targets that can be used in the process;methods for partitioning nucleic acids within a candidate mixture; andmethods for amplifying partitioned nucleic acids to generate enrichedcandidate mixture. The SELEX Patent Applications also describe ligandsobtained to a number of target species, including both protein targetswhere the protein is and is not a nucleic acid binding protein.

The methods described herein and the nucleic acid ligands identified bysuch methods are useful for both therapeutic and diagnostic purposes.Therapeutic uses include the treatment or prevention of diseases ormedical conditions in human patients. Therapeutic uses may also includeveterinary applications.

Diagnostic utilization may include both in vivo or in vitro diagnosticapplications. The SELEX method generally, and the specific adaptationsof the method taught and claimed herein specifically, are particularlysuited for diagnostic applications. SELEX identifies nucleic acidligands that are able to bind targets with high affinity and withsurprising specificity. These characteristics are, of course, thedesired properties one skilled in the art would seek in a diagnosticligand.

The nucleic acid ligands of the present invention may be routinelyadapted for diagnostic purposes according to any number of techniquesemployed by those skilled in the art. Diagnostic agents need only beable to allow the user to identify the presence of a given target at aparticular locale or concentration. Simply the ability to form bindingpairs with the target may be sufficient to trigger a positive signal fordiagnostic purposes. Those skilled in the art would also be able toadapt any nucleic acid ligand by procedures known in the art toincorporate a labeling tag in order to track the presence of suchligand. Such a tag could be used in a number of diagnostic procedures.The nucleic acid ligands to TGFβ described heroin may specifically beused for identification of the TGFβ protein.

SELEX provides high affinity ligands of a target molecule. Thisrepresents a singular achievement that is unprecedented in the field ofnucleic acids research. The present invention applies the SELEXprocedure to the specific target of TGFβ1. In the Example section below,the experimental parameters used to isolate and identify the nucleicacid ligands to TGFβ1 are described.

In order to produce nucleic acids desirable for use as a pharmaceutical,it is preferred that the nucleic acid ligand (1) binds to the target ina manner capable of achieving the desired effect on the target; (2) beas small as possible to obtain the desired effect; (3) be as stable aspossible; and (4) be a specific ligand to the chosen target. In mostsituations, it is preferred that the nucleic acid ligand have thehighest possible affinity to the target.

In co-pending and commonly assigned U.S. patent application Ser. No.07/964,624, filed Oct. 21, 1992 ('624), now U.S. Pat. No. 5,496,938,methods are described for obtaining improved nucleic acid ligands afterSELEX has been performed. The '624 application, entitled Methods ofProducing Nucleic Acid Ligands, is specifically incorporated herein byreference.

In the present invention, SELEX experiments were performed in order toidentify RNA and DNA ligands with specific high affinity for TGF62 fromdegenerate libraries containing 40 or 60 random positions (40N or 60N)(Tables 1 and 5) (SEQ ID. NOS: 1-3, 43, 46, 49, 52). This inventionincludes the specific RNA ligands to TGFβ1 shown in Table 3 (SEQ ID NOS:12-42), identified by the methods described in Examples 1 and 2. Thisinvention further includes RNA ligands to TGFβ1 which inhibit TGFβ1function, presumably by inhibiting the interaction or TGFβ1 with itsreceptor. This invention includes the specific ssDNA ligands to TGFβ1shown in Table 6 (SEQ ID NOS: 55-89) identified by the methods describedin Examples 5 and 6. The scope of the ligands covered by this inventionextends to all nucleic acid ligands of TGFβ, modified and unmodified,identified according to the SELEX procedure. More specifically, thisinvention includes nucleic acid sequences that are substantiallyhomologous to the ligands shown in Tables 3 and 6 (SEQ ID NOS: 12-42;35-89). By substantially homologous it is meant a degree of primarysequence homology in excess of 70%, most preferably in excess of 80%. Areview of the sequence homologies of the ligands of TGFβ shown in Tables3 and 6 (SEQ ID NOS: 12-42; 55-89) shows that some sequences with littleor no primary homology may have substantially the same ability to bindTGFβ. For these reasons, this invention also includes nucleic acidligands that have substantially the same structure and ability to bindTGFβ as the nucleic acid ligands shown in Tables 3 and 6 (SEQ ID NOS:12-42; 55-89). Substantially the same ability to bind TGFβ means thatthe affinity is within one or two orders of magnitude of the affinity ofthe ligands described herein. It is well within the skill of those ofordinary skill in the art to determine whether a givensequence--substantially homologous to those specifically describedherein--has substantially the same ability to bind TGFβ.

This invention also includes the ligands as described above, whereincertain chemical modifications are made in order to increase the in vivostability of the ligand or to enhance or mediate the delivery of theligand. Examples of such modifications include chemical substitutions atthe sugar and/or phosphate and/or base positions of a given nucleic acidsequence. See, e.g., U.S. patent application Ser. No. 08/117,991, filedSep. 9, 1993, entitled High Affinity Nucleic Acid Ligands ContainingModified Nucleotides which is specifically incorporated heroin byreference. Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process.

As described above, because of their ability to selectively bind TGFβ,the nucleic acid ligands to TGFβ described herein are useful aspharmaceuticals. This invention, therefore, also includes a method fortreating TGFβ-mediated pathological conditions by administration of anucleic acid ligand capable of binding to TGFβ.

Therapeutic compositions of the nucleic acid ligands may be administeredparenterally by injection, although other effective administrationforms, such as intraarticular injection, inhalant mists, orally activeformulations, transdermal iontophoresis or suppositories, are alsoenvisioned. One preferred carrier is physiological saline solution, butit is contemplated that other pharmaceutically acceptable carriers mayalso be used. In one preferred embodiment, it is envisioned that thecarrier and the ligand constitute a physiologically-compatible, slowrelease formulation. The primary solvent in such a carrier may be eitheraqueous or non-aqueous in nature. In addition, the carrier may containother pharmacologically-acceptable excipients for modifying ormaintaining the pH, osmolarity, viscosity, clarity, color, sterility,stability, rate of dissolution, or odor of the formulation. Similarly,the carrier may contain still other pharmacologically-acceptableexcipients for modifying or maintaining the stability, rate ofdissolution, release, or absorption of the ligand. Such excipients arethose substances usually and customarily employed to formulate dosagesfor parental administration in either unit dose or multi-dose form.

Once the therapeutic composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready to use form or requiring reconstitution immediately prior toadministration. The manner of administering formulations containingnucleic acid ligands for systemic delivery may be via subcutaneous,intramuscular, intravenous, intranasal or vaginal or rectal suppository.

The following Examples are provided to explain and illustrate thepresent invention and are not intended to be limiting of the invention.Examples 1-4 describe initial experiments to identify RNA with specifichigh affinity for TGFβ. Example 1 describes the various materials andexperimental procedures used in Examples 2-4. Example 2 describes arepresentative method for identifying RNA ligands by the SELEX methodwhich bind TGFβ1. Example 3 describes the affinities the ligands havefor TGFβ1 and demonstrates that the ligands are capable of inhibitingthe function of TGFβ1, presumably by inhibiting the interaction of TGFβ1with its receptor. Example 4 describes which regions of the ligands arebelieved to be necessary for TGFβ1 binding and inhibition of TGFβ1receptor binding. Example 5 describes another representative method foridentifying RNA and DNA ligands by the SELEX method which bind TGFβ1.Example 6 reports on the binding analysis, bioassay, and sequences of assDNA SELEX library.

EXAMPLES Example 1

EXPERIMENTAL PROCEDURES

This example provides the general procedures followed and incorporatedin Examples 2-4.

A. Materials.

Human recombinant TGFβ1 used in this SELEX procedure was acquired fromGenentech. Human recombinant TGFβ1 can also be purchased from R&Dsystems, Minneapolis, Minn., USA.

Biotinylated TGFβ1 was prepared by reacting TGFβ1 at 3.6 μM with an 11fold molar excess of sulfo-NHS-biotin (Pierce, Rockford, Ill., USA) in50 mM NaHCO₃ for 3 hr. in an ice bath. The reaction was acidified with0.036 volumes of 10% acetic acid and applied to a 40 mg. Vydac (TheSeparations Group, Hesperia, Calif., USA) reverse phase column made in asiliconized pipet tip to separate unreacted biotin from biotinylatedTGFβ1. The column was prewashed with 200 μl ethanol followed by 200 μl1% acetic acid, the biotinylation reaction was applied, free biotin waswashed through with 200 μl of 50 mM sodium acetate pH 5.5, followed by200 μl of 20% acetonitrile and finally eluted with 200 μl of 60%acetonitrile. The sample was lyophilized and resuspended in 50 mM sodiumacetate pH 5.0 at 40 μM and stored at 4° C. The TGFβ1 was spiked with100,000 cpm iodinated TGFβ1 in order to follow recovery and to assessthe success of the biotinylation reaction by measuring the fraction ofthe radioactivity that would bind to streptavidin coated agarose beads(Pierce) before and after biotinylation. An aliquot of the TGFβ1 beforeand after biotinylation was subjected to analytical reverse phasechromatography. The biotinylated TGFβ1 substantially ran as a singlepeak which was retarded with respect to the unbiotinylated TGFβ. A smallamount (5%) of unreacted TGFβ1 could be detected. The efficiency ofbinding of the iodinated, biotinylated TGFβ1 to streptavidin (SA)agarose beads (30 μl) was 30% under the binding conditions used forSELEX partitioning.

Iodinated TGFβ1 was prepared by the lactoperoxidase method (50 mM sodiumphosphate pH 7.3, 0.16% glucose) with BioRad Enzymo beads (BioRad,Richmond, Calif. USA) and the bound iodine separated from the freeiodine by gel filtration on G25 Sephadex in 50 mM sodium acetate 0.01%Tween.

The mink lung cell line expressing the luciferase reporter gene underthe control of PAI 1 promoter (Abe et al. (1994) Anal. Biochem.216:276-284) was a gift from Dr. Dan Rifkin (Department of Cell Biology,New York Medical Center, N.Y. 10016). Luciferase was assayed by reagentspurchased from Analytical Luminescence Laboratory, San Diego, Calif.USA.

2'-NH₂ modified CTP and UTP were prepared according to the method ofPieken et al. (1991. Science 253:314-317). DNA oligonucleotides weresynthesized using standard procedures either at NeXstar Pharmaceuticals,Inc. (Boulder, Colo, USA) or by Operon Technologies (Alameda, Calif.USA). All other reagents and chemicals were purchased from standardcommercial sources and sources have been indicated.

B. SELEX procedure

SELEX ligands that bind to TGFβ1 were derived essentially as describedin U.S. Pat. No. 5,270,163 (see also Tuerk and Gold (1990) Science249:505-510). To generate the starting pool of PCR template, PCR productfrom twenty separate PCR reactions each containing 16.1 pmol ofunpurified, single stranded DNA (at least a total of 2×10¹² to 2×10¹³different molecules) were pooled before the first transcription. PCRconditions were 50 mM KCl, 10 mM Tris-HCL, pH 9, 0.1% Triton-X100, 1.5mM MgCl₂, 0.2 mM of each dATP, dCTP, dGTP, and dTTP, 2 μM each primerand 0.075 units/μl of Taq DNA polymerase, 100 μl per reaction in asiliconized microfuge tube. All PCR cycles took advantage of hot startusing Ampliwax (Perk and Elmer, Norwalk, Conn., USA). Duration of theinitial PCR was 10 cycles; a PCR cycle was 94° C.-1', 52° C.-1', 72°C.-2'. An initial denaturation was 94° C. for 4' and the final extensionat 72° C. for 5'. PCR reactions were combined, phenol/chloroformextracted, and isopropanol precipitated (2.0M ammonium acetate, 50%isopropanol) to remove primers.

Transcription reactions contained 200 nM DNA, 0.9 mM GTP, 0.9 mM 2'-NH₂-UTP, 0.9 mM 2'-NH₂ -CTP, 0.5 mM ATP, 87 mM Tris-HCl pH 8.0, 17 mMMgCl₂, 4.4 mM spermidine, 22 mM DTT, 100 μg/ml acetylated BSA (Promega,Madison, Wis., USA) and 4 units/μl T7 RNA polymerase. (2'-F-UTP and2'-F-CTP (United States Biochemical, Cleveland, Ohio, USA) were used at3.0 mM, whereas UTP and CTP were used at 0.9 mM each). Transcriptionreactions were incubated overnight at 28° C. (at least 10 hours). Aftertranscription the template was digested by addition of 2 μl RQ1 Dnase(Promega) for 15' at 28° C., and then extracted with phenol/CHCl₃,followed by three ethanol precipitations from ammonium acetate (3.9Mammonium acetate, 72% ethanol).

The RNA molecules were incubated with TGFβ1 bound to SA agarose beads asdescribed below in Krebs-Ringer solution (KR) (120 mM NaCl, 4.8 mM KCl,10 mM Na phosphate buffer pH 7.4, 1.2 mM MgSO₄, 2.6 mM CaCl₂) modifiedto include 20 mM Na-Hepes pH 7.5 and 0.2% Triton X100 (Pierce). Thisbuffer is referred to as KRHT. TGFβ1-RNA complexes were separated fromunbound RNA by washing the beads. Recovery of the selected 2'-NH₂ or Fpyrimidine modified RNA from the agarose beads required guanidinethiocyanate extraction (5M GnSCN, 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.0,0.1M beta mercaptoethanol) or from Seradyne SA coated beads by 2% SDS(0.1M Tris-HCl pH 7.5, 50 mM NaCl,1 mM Na₂ EDTA, 2% SDS, 1.5mM DTT).Regular 2'-OH RNA was easily recovered under less harsh conditions withthe same buffer used for the Seradyne beads containing only 0.2% SDS.After extraction and precipitation to purify and concentrate the RNA,the sample was reverse transcribed with a cloned MMLV RT with the RNaseH sequence deleted. The reaction contained less than or equal to 16 nMRNA, 10 μM 3' primer, 50 mM Tris-HCL pH 8.3, 75 mM KCl,5 mM MgCl₂, 10 mMDTT, 0.5 mM dNTP's. Prior to addition of buffer the RNA and the palmerwere boiled together. After addition of buffer and salts the reactionwas annealed for 10' at 28° C. before addition of 600 units ofSuperscript reverse transcriptase (Bethesda Research Labs, Gaithersburg,Md., USA) and synthesis at 50° C. for 1 hour.

PCR amplification of this cDNA (<1 pmol) resulted in approximately 250pmol double-stranded DNA, of this 40 pmols was transcribed and used toinitiate the next round of SELEX.

C. Partitioning Method for SELEX.

2.5 pmols biotinylated TGFβ1 were bound to 30 μl SA agarose beads(Pierce) in 200 μl KRHT. The mixture was incubated on a rotator at 37°C. for 15 to 30 minutes. The beads were washed three times bycentrifugation and resuspension in 200 μl cold KRHT to remove unboundTGFβ1, and resuspended in a final volume of 500 μl KRHT. RNA containing2'-NH₂ pyrimidines was heated at 70° C. for three minutes (RNAscontaining 2'-OH or 2'-F pyrimidines were heated at 95° C.) and dilutedinto KRHT containing TGFβ1 bound to SA beads. The final concentration ofRNA is 1 μM and the TGFβ1 was 5 nM. Binding occurs with rotation at 37°C. for 30 minutes. Beads were washed by centrifugation and resuspensionthree times with 200 μl binding buffer to remove unbound RNA. RNA waseluted from the beads as described above.

D. Binding assays

Two binding assays for ligands to TGFβ1 gave equivalent results wherevertested. In the SA bead assay the biotinylated TGFβ1 was serially dilutedin KRHT in polypropylene tubes (Linbro, ICN, Irvine, Calif., USA) andbound to the beads as described above. After unbound TGFβ1 was washedaway, trace quantities of ³² P -labeled RNA(<0.1 nM) were added to eachtube and vortexed to mix. After static incubation at 37° C. for 30minutes, the unbound RNA was removed by washing three times with 200 μlof KRHT.

In the nitrocellulose filter binding assay, TGFβ1 was serially dilutedin KRH containing 0.1% defatted BSA (Fluka radioimmunoassay grade,Fluka, Hauppauge, N.Y., USA) as carrier instead of Triton X-100.Incubation with RNA tracer was as above. Samples were pipetted with amultiwell pipettor onto a multiwell manifold holding a sheet of wetBioRad 0.45 micron nitrocellulose, aspirated, and washed three timeswith 200 μl KRH (containing no BSA). The filters were air dried andcounted in a liquid scintillation counter (Beckmann Instruments, PaloAlto, Calif.)

The equation used to fit the binding of ligands to TGFβ1 describes thebinding of a ligand to a receptor (in this case TGFβ1) that follows thelaws of mass action and for which there is a single binding site:Y=Bmax*X/(Kd+X): where Y is the fraction of the ligand bound, B_(max) isthe maximum fraction of the ligand bound, X is the concentration ofTGFβ1 and Kd is the dissociation constant of TGFβ1 and the ligand. Datapoints were fit by nonlinear regression using the computer programGraphpad Prism (Graphpad Software, San Diego, Calif.). The algorithmminimized the sum of the squares of the actual distance of the pointsfrom the curve. Convergence was reached when two consecutive iterationschanged the sum-of-squares by less than 0.01%.

E. Cloning and Sequencing

SELEX experiments are described in Table 2. Primers for SELEXexperiments 1 and 2 shown in Table 1 contain recognition sites for therestriction endonuclcases SacI (5' primer T7SacBam; SEQ ID NO: 7) andXbaI (3' primer 3XH; SEQ ID NO:9). PCR products from SELEX experiments 1and 2 were cloned directionally into SacI/XbaI digested pGem 9zf(Promega). 5' primer T7SB2N (SEQ ID NO: 8) and 3' primer 3XH (SEQ ID NO:9) (Table 1) were used for SELEX experiments 3-9. PCR products fromSELEX experiments 3-9 were cloned directionally into the BamH1/XbaI siteof a modified pGem9zf :BamH1 cloning vector. The BamH1 site wasengineered into pGem9zf in the following way. A clone isolated fromlibrary 2 (lib2-6-2) that did not bind to TGFβ1 (sequence not shown) wasdigested with BaMH1 and XbaI. The sequence flanking the cloning site ofthe modified pGem9zf vector is shown in Table 1 (SEQ ID NOS: 10-11).

After digestion of the plasmid with restriction endonuclease anddephosphorylation with CIP (calf intestinal phosphatase), vectors weregel purified. Inserts were ligated and recombinant plasmids weretransformed into E. coli strain DH10B (Bethesda Research Labs). PlasmidDNA was prepared by alkaline lysis, mini prep procedure. Twenty-twoclones representing 9 unique sequences were sequenced at random fromlibraries 1 and 2. 50 clones were sequenced from libraries 3-9 using asingle dideoxy G reaction (called G track). The sequencing ladders werecompared and organized for similarities. Selected clones from eachfamily were chosen for complete sequence analysis. TGFβ1 binding assayswere performed on transcripts representing different G sequences in eachlibrary. Out of a total of 140 binding assays, 27 ligands bound with aKd less than 10 nM, and 21 of these were sequenced. Clones weresequenced with the Sequenase sequencing kit (United States BiochemicalCorporation, Cleveland, Ohio).

F. Ligand Truncation

Truncation experiments were carded out to determine the minimal sequencenecessary for high affinity binding of the RNA ligands to TGFβ1. For 3'boundary determination, RNA ligands were 5' end-labeled with γ-³² P-ATPusing T4 polynucleotide kinase. 5' boundaries were established with 3'end-labeled ligands using α-³² P-pCp and T4 RNA ligase. After partialalkaline hydrolysis, radiolabeled RNA ligands were incubated with TGFβ1at concentrations ranging from 1 nM to 50 nM and protein-bound RNA wasseparated by nitrocellulose partitioning. RNA truncates were analyzed ona high-resolution denaturing polyacrylamide gel. A ladder ofradioactively labeled ligands terminating with G-residues was generatedby partial RNase T1 digestion and was used as markers.

G. Inhibition of TGFβ1 function

TGFβ1 signal transduction begins with binding to a cell surface receptorand results in the induction of transcription of a variety of genes. Oneof these genes is Pai1. The TGFβ1 assay utilizes the mink lungepithelial cell (MLEC) carrying the luciferase reporter gene fused tothe Pai 1 promoter. The MLEC has TGFβ1 receptors on its cell surface.Thus one can measure the response of the cells to TGFβ1 and theeffective concentration of TGFβ1 in the culture media by measuring theluciferase enzyme activity after a period of induction.

Mink lung epithelial cells (MLEC) carrying the Pai1/luc construct weremaintained in DME containing 10% fetal bovine serum and 400 μg/ml G418.MLEC-Pai1/luc cells were plated at 3.2×10⁴ cells per well in a 96 wellFalcon plate, in 100 μl of DME+10% fetal bovine serum overnight. Mediawas made from autoclaved water. The cells were washed three times (100μl) in serum free DME plus Solution A (1:1). Solution A is 30 mM HepespH 7.6, 10 mM glucose, 3.0 mM KCl, 131 mM NaCl, 1.0 mM disodiumphosphate. Samples (100 μl) were added in DME containing 20 mM Hepes pH7.5, and 0.1% BSA (Fluka, radioimmunoassay grade). All samples were intriplicate. After six hours at 37° C. in a 5% CO₂ incubator the mediawas removed and cells were washed three times (100 μl each) in cold PBS.Lysis buffer (75 μl) (Analytical Luminescence Laboratory) was added andthe plates incubated on ice for 20'. The plates were sealed and frozenat -80° C. until assayed. Samples (25 μl) were assayed for luciferaseactivity with the Enhanced Luciferase Assay Kit from AnalyticalLuminescence Laboratory (San Diego, Calif., USA) according to themanufacturers instructions using the Berthold Microlumat LB96Pluminometer. Luminescence is reproducibly a function of TGFβ1concentration added to the media.

Ligands tested for inhibition of TGFβ1 activity were tested at a minimumof five concentrations. The ligands were serially diluted in DME, 20 mMHepes pH 7.5, 0.1% Fluka BSA in polypropylene tubes and an equal volumeof media containing 12 pM TGFβ1 was added to each tube, vortexed andtransferred to the cells without further incubation. From the TGFβ1standard curve included on every plate the effective concentration ofTGFβ1 in the presence of the inhibitory ligands was determined by thereduction in luminescence measured. Some ligands were tested at both 3pM and 6 pM TGFβ1 with the same results. To determine the IC₅₀ (theconcentration of SELEX ligand necessary to reduce the TGFβ1 activity50%), the five values obtained for each ligand were plotted and thevalue at 50% inhibition was determined graphically using Graphpad Prismassuming a hyperbolic fit of the data and using non-linear regression.

Example 2

RNA LIGANDS TO TGFβ1

A. SELEX experiments

In order to generate RNA ligands to TGFβ1, nine SELEX experiments, assummarized in Table 2, were performed using the methods described inExample 1. As shown in Table 1, the RNA pools differ in the number ofrandom bases present in the central portion of the molecules: 40nucleotides in the 40N6 (SEQ ID NO: 2) SELEX and 64 nucleotides in the64N6 and lib2-6-1RN6 (SEQ ID NOS: 1, 3) SELEX experiment. Since the goalwas to select RNA ligands that not only bound to TGFβ1 but also blockedreceptor binding, the large random region (64N) was chosen. In twoexperiments, a shorter random region (40N) was also included. Ligands toTGFβ1 were very rare with 40N and were qualitatively the same as the64N6 ligands selected.

The sequences of clones from the SELEX experiments are shown in Table 3(SEQ ID NOS: 12-42). The pyrimidines used in the various SELEXexperiments differed at the 2' position of the sugar (Table 2). In thefirst two SELEX experiments, ligands were evolved as 2'-OH pyrimidines.Ligands were post-SELEX modified with 2'-NH₂ or 2'-F- substitutedpyrimidines to see if they retained TGFβ1 binding. Since the 2'substitutions rendered the ligands resistant to RNase A they were alsotested in the cell culture assay for inhibition of TGFβ1 activity. Onesuch ligand lib2-6-1 (Group D, Table 3) (SEQ ID NO: 35) when substitutedwith 2-NH₂ -UTP and 2'-F-CTP was shown to inhibit TGFβ1 receptormediated activity. To select more ligands, six more independent SELEXexperiments (lib3-7 and lib9) were performed using the 2'-F and 2'-NH₂-substituted pyrimidines during the evolution process. In experimentlib8 the biologically active clone lib2-6-1 (SEQ ID NO: 35) wasrandomized and subjected to re-selection to see if the binding andinhibition behavior of the clone could be improved. Lib8 was evolved asa 2'-OH pyrimidine RNA. In some cases, post -SELEX modification of TGFβ1ligands derived from experiments 3-9 were performed, eg. to determine ifa ligand evolved with 2'-F pyrimidine substitutions would also bind with2'-NH₂ substitutions.

Each starting pool for a SELEX experiment contained 3×10¹⁴ RNA molecules(500 pmol). The affinity of the starting RNA for TGFβ1 was estimated tobe greater than 50 mM. After 4 rounds of SELEX, the affinities of theevolving pools had improved to approximately 10 nM and did not shiftsignificantly in subsequent rounds. RNA was bulk sequenced and found tobe non-random and cloned.

Lib1 took 20 rounds to evolve since optimum concentrations of TGFβ1 werenot used until round 15 and libraries 5, 6 and 7 took longer to evolvebecause optimum conditions for recovery of bound ligands during thepartitioning step in SELEX were not introduced until round 8. OptimumTGFβ1 concentrations and partitioning conditions are described inExample 1.

B. RNA Sequences

Many clones in a SELEX library are identical or similar in sequence. Thelibraries were screened by G track and only representatives of each Gtrack type were tested in a binding assay. The binding assay was fivepoints (16.5 nM, 5.5 nM, 1.8 nM, 0.6 nM, and 0.2 nM) and could detectonly those SELEX clones with a Kd less than or equal to 10 nM. RNAligands that bound well (Kd<10 nM) in the binding assay were sequenced.The sequences were inspected by eye and analyzed using computer programswhich perform alignments and fold nucleic acid sequences to predictregions of secondary structure. Ligands were classified into five groups(A, B, C, D, and orphans) by sequence homology. Each group hascharacteristic allowable 2' substitutions.

58 clones were identified by G track from 7 separate SELEX experimentsto belong to group A ligands (Table 3) (SEQ ID NOS: 12-42). 15 cloneswere sequenced; 13 were similar but not identical, whereas 3 clones,lib3-13 (SEQ ID NO: 12), lib5-6 and lib5-13, were identical. Group Aligands were recovered from seven of the eight SELEX libraries whichincluded libraries evolved as 2'-NH₂, 2'-OH or 2'-F-substitutedpyrimidines as well as a library evolved as 2'-F-UTP, 2'-NH₂ -CTP. PostSELEX modification indicates that 2'-NH₂ -UTP, 2'-F-CTP does not disruptbinding of lib8-9 to TGFβ1, thus the structure of Group A ligandsappears to not require a specific 2' moiety on the pyrimidine sugar inorder to maintain binding.

Group B ligands bind both as 2'-NH₂ and 2'-F pyrimidine substituted RNA.28 Group B clones were detected by G track analysis from 3 libraries.Two of the libraries were evolved as 2'-NH₂ and one as 2'-F library.Four clones were sequenced, two were identical (lib5-47 and lib4-12)(SEQID NO: 28). An internal deletion can occur in group B, as does in lib3-44 (SEQ ID NO: 29). The 40N orphan, lib3-42 was placed in Group B onthe basis of secondary structure. The internal deletion in lib3-44, thebinding affinity, the bioactivity and boundary experiments all supportthe placement of lib3-42 in this group.

Group C ligands bind as 2'-OH or 2'-F ligands as expected, since membersof this group were evolved as 2'-OH ligands in lib 1 and as 2'-Fpyrimidine substituted ligands in lib 6. Lib 1-20-3 was post SELEXmodified and as 2'-F derivative. Lib 1-20-3 (SEQ ID NO: 32) did not bindwith 2'-NH₂ pyrimidines incorporated.

Group D ligand, lib2-6-1 (SEQ ID NO: 35) was isolated after 2'-OH SELEXbut was post SELEX modified and binds well as a 2'-NH₂ -UTP and 2'-F-CTPpyrimidine derivative. Lib2-6-1 does not bind well to TGFβ1 with 2'-NH₂,2'-F or 2'F-UTP, 2'-NH₂ -CTP -substituted pyrimidines. Variants of GroupD were only reselected in two other SELEX experiments, lib8, a 2'-OHlibrary, and lib 9, a 2'-NH₂ -UTP, 2'-F CTP library, supporting theobservation that there is specificity for the 2' pyrimidine position inthis ligand.

The group labeled orphans are not homologous to each other and novariant sequences for these ligands have been determined. G trackindicates that eight 40N clones similar to lib3-45 (SEQ ID NO: 39) wereisolated from two libraries. Two of the eight were sequenced and areidentical. Lib3-45 binds whether it contains 2'-NH₂ or 2'-F substitutedpyrimidines or the 2'-F-UTP, 2'-NH₂ -CTP combination. Libl-20-5 (SEQ IDNO: 40) isolated as a 2'-OH ligand binds as a 2'-F, whereas libl-20-12(SEQ ID NO: 41) and lib2-6-8 (SEQ ID NO: 42) bind well only as 2'-OHpyrimidines and will not tolerate 2'-NH₂ or 2'-F post SELEXmodifications.

As it was unusual that similar sequences were obtained from differentselex experiments containing different modifications, another set ofselex experiments was performed in search of RNA and ssDNA ligands toTGFβ1 as described in examples 5 and 6 infra.

Example 3

INHIBITION OF TGFβ1 RECEPTOR BINDING

The Kds and B_(max) values reported in Table 4 for Group A ligands arefor the 2'-NH₂ substituted version of the ligand unless otherwise noted.B_(max) for the Group A ligands was 0.38±0.12 (n=14) which is inagreement with the measured retention of TGFβ1 on the nitrocellulosefilters. The Kd's for Group A ligands were all similar, 2.2±1.1 nM(n=14). Where measured nitrocellulose and SA agarose bead binding assaysgave equivalent results.

The IC₅₀ 's in Table 4 for Group A ligands were all tested with the2'-NH₂ pyrimidine substituted ligands except where indicated. 2'-NH₂ligands were used in the tissue culture bio-assay since they exhibitedthe greatest stability under the conditions of the bio-assay. Five outof ten Group A ligands tested inhibited TGFβ1 receptor activity. IC₅₀values for the inhibitors were typically 25 fold above the Kd for TGFβ1.The data are reproducible; the Kd for ligand lib3-13 was 0.83±0.11 nM(n=3) and the IC₅₀ for lib3-13 was 25±14 nM (n=4). RNA concentrations inthe bioassays are all estimates based on an assumed extinctioncoefficient and 100% purity of the ligand. The RNA concentrations may,therefore, be overestimated during the bio-assay which in turn wouldoverestimate the IC₅₀.

Another five Group A ligands did not inhibit TGFβ receptor bindingactivity. One obvious difference between the non-bioactive ligands,lib2-6-4 (SEQ ID NO:20), lib5-48 (SEQ ID NO: 19), and lib6-23(SEQ IDNO:21), and the bioactive ligands is the substitution at nucleotide 72.Lib7-21 (SEQ ID NO: 23) and lib7-43 (SEQ ID NO: 24) were tested as2'-F-UTP, 2'-NH₂ -CTP ligands for bio-activity. These ligands were notbioactive despite their high affinity to TGFβ. In conclusion, bindingand bioactivity are separable functions of the TGFβ Group A ligands.

Group B ligands have different binding properties than Group A ligands(Table 4). Both the Kd (0.63±0.5 nM, n=4) and B_(max) (0.14±0.04, n=4)are lower for Group B ligands. One Group B inhibitor, lib4-12 (SEQ IDNo: 28), actually appears to stimulate TGFβ1 activity in the tissueculture bio-assay at low concentrations. The basis of this mixedagonist/antagonist behavior has not been determined. The best inhibitorin this group, lib3-42 (SEQ ID NO: 30) has an IC₅₀ of 22 nM and had noagonist behavior over the concentration ranges tested.

Group C ligands were tested as 2'-F derivatives and were not bio-active.Neither was the 2'-F orphans lib1-20-5 (SEQ ID NO: 40). The 2'-NH₂, 40Norphan, lib3-45 (SEQ ID NO: 39) is an example of another ligand withhigh affinity for TGFβ1 and no ability to inhibit TGFβ1 receptorbinding.

Group D ligands were tested in the bio-assay as 2'-NH₂ -UTP, 2'-F-CTPderivatives. Both lib2-6-1 (SEQ ID NO: 35) and the truncated versionlib2-6-1-81 (SEQ ID NO: 36) can inhibit TGFβ1 receptor binding; however,a single mutation from a C to a G at position 53 decreases bio-activityin clone lib8-23 (SEQ ID NO: 37). Similarly a 2 base pair deletion inclone lib6-30 (SEQ ID NO: 34) at positions corresponding to nucleotides67 and 68 in lib2-6-1 (SEQ ID NO: 35) increases binding by 10 fold buteliminates bio-activity.

Lib2-6-1 (SEQ ID NO: 35) was shown to be fully effective only againstTGFβ1 and not TGFβ2 and TGFβ3. Lib2-6-1 was biologically active in thepresence of 10% horse serum in the cell culture medium in addition tothe 0.1% BSA. Thus the ligand demonstrates specificity towards TGFβ1which is not interferred with by the presence of the horse serum in thisassay. The biggest indication that the inhibition of TGFβ1 receptorbinding is a specific phenomenon is the fact that not all TGFβ1 ligandsblock receptor binding, but the ones that do, do so reproducibly. Thereare no examples of ligands that do not bind to TGFβ1 blocking TGFβ1receptor binding activity.

In summary, RNA ligands that can block TGFβ1 receptor binding are asubset of ligands. Binding is necessary but not sufficient forbio-activity. Roughly 50% of the high affinity ligands tested wereinhibitors. Of the inhibitors, 30% were good inhibitors (IC₅₀ <25 nM).

Example 4

BOUNDARY ANALYSIS

Truncation experiments were done on a number of TGFβ1ligands todetermine the nucleotides essential for binding. Group A ligands,lib3-13 (SEQ ID NO: 12) and lib8-9 (SEQ ID NO: 16), were truncated withconsistent results. The fragment lib3-13-79 binds to TGFβ1, thus none ofthe nucleotides 3' to nucleotide 79 in lib3-13 am essential for binding.Similarly when all nucleotides 5' to nucleotide 38 are deleted theremaining fragment, lib3-13-(38-123) can still bind to TGFβ1. The 5'boundary is in agreement with the sequence lib6-23 (SEQ ID NO: 21),which has a deletion corresponding to nucleotides 19-36 of lib3-13 (SEQID NO: 25), and still binds to TGFβ1. Thus, all high affinity bindingdeterminants for Group A clones may lie wholly within the random regionand may correspond to a 42 nucleotide fragment, lib3-13-(38-79). ManyGroup A ligands contain deletions or substitutions within the predictedessential binding domain, in the region corresponding tolib3-13-(72-81). The deletion and substitution in lib4-32 have no effecton its 3' boundary which corresponds to lib3-13 nucleotide 80. Thus, the3' boundary is probably correct and the alterations in nucleotidesequence 72-81 are ones that do not significantly alter the nucleic acidstructure required for binding. Mutations in this region, most notablynucleotide 72 may, however, modify the ability of the ligand to blockTGFβ1 receptor binding as noted earlier.

Boundary analysis of the 3' end of Group B ligand, lib4-12 (SEQ ID NO:28), predicts that nothing beyond nucleotide 72 is required for TGFβ1binding. When the 5' boundary of lib4-12 was determined, all but thefirst three nucleotides were required for binding, indicating that the5' constant region is an essential part of the ligand at least when theboundary of the full length ligand was determined. Assuming that ligandlib3-44 (SEQ ID NO: 29) has a similar binding determinant as lib4-12(SEQ ID NO: 28), we can also conclude that nucleotides 37-46 of lib4-12are not required for binding since these are deleted in lib3-44 andlib3-42 (SEQ ID NO: 30).

The 3' constant region is not necessary for binding in Group C and Dligands. Both ligand types bind without the 3' nucleotides in theconstant region. Lib1-20-3-82, an 82 nucleotide truncated version oflib1-20-3, binds as well as the full length lib1-20-3. Likewise bindingand bioactivity of lib2-6-1 (SEQ ID NO: 35) is unaffected by the 3'truncation found in lib2-6-1-81 (SEQ ID NO: 36).

Example 5

EXPERIMENTAL PROCEDURES

In the preferred embodiment, a second set of SELEX experiments wasperformed in search of RNA and DNA ligands with specific high affinityfor TGFβ1 from degenerate libraries containing 40 random positions(40N). This Example provides the general procedures followed andincorporated in Example 6.

A. Materials

M-MLV superscript reverse transcriptase was purchased from Gibco BRL(Gaithersburg, Md.). T7 RNA polymerase was purified according tostandard procedures at NeXstar Pharmaceuticals, Inc. (Boulder, Colo.).Taq DNA polymerase (Amplitaq) was from Perkin Elmer/Cetus (Richmond,Calif.). T4 polynucleotide kinase, DNA polymerase (Klenow fragment), andalkaline phosphatase were purchased from New England Biolabs, Inc.(Beverly, Mass.). The 2'-amino substituted nucleotide triphosphatesamino-UTP and amino-CTP were synthesized according to standardprocedures at NeXstar Pharmaceuticals, Inc. (Boulder, Colo.). Otherreagents used in this work were of the highest quality obtainable.

B. Nucleic Acids

RNAs were synthesized by in vitro transcription using double-strandedDNA oligonucleotides and T7 RNA polymerase. DNA oligonucleotides (Table5) were purchased from Operon, Inc. (Alameda, Calif.) and purified by 6%preparative polyacrylamide gel electrophoresis. PCR amplification wasperformed in 50 mM KCl, 10 mM Tris-HCl (pH 8.6), 2.5 mM MgCl₂, 170 mg/mLBSA, and dNTPs (present at 1 mM each). Taq DNA polymerase was used at100 units per 0. 1 mL reaction, and the 5'- and 3'-primers were presentat 1 mM. Transcription was performed in 40 mM NaCl, 10 mMdithiothreitol, 50 mM Tris-acetate (pH 8.0), 8 mM magnesium acetate, 2mM spermidine, and 2 mM NTP. T7 RNA polymerase was present at 1 unit/mL.The reaction was incubated at 28 degrees for 16 hours and then treatedwith 20 units of DNAse I for an additional 10 min at 37 degrees. Thereaction was stopped by the addition of one half volume of loadingbuffer (93% formamide, 10 mM EDTA, pH 8.0) and heated to 95 degrees for3 min prior to electrophoresis on a 6% polyacrylamide/8M urea denaturinggel. The RNA transcript was visualized by UV shadowing and was excisedfrom the gel and eluted into TE buffer (10 mM Tris-acetate pH 8.0, 2 mMEDTA). The RNA transcript was ethanol precipitated, dried under vacuum,and redissolved in distilled H₂ O. The concentration of RNA as well assingle-stranded DNA was quantified by measuring the A₂₆₀ and assumingthat 1 A₂₆₀ unit equaled 40 mg/mL and 33 mg/mL, respectively.

C. Evolution of High-Affinity Ligands

SELEX ligands that bind to TGFβ1 were derived essentially as describedin U.S. Pat. No. 5,270,163 (see also Tuerk and Gold (1990) Science249:505-510) using the oligonucleotides illustrated in Table 5 (SEQ IDNOS: 43-54). The DNA templates contained a 40-nucleotide (40N) variablesequence generated by mixed-nucleotide DNA synthesis, as well as 5'- and3'-fixed sequences, necessary for PCR amplification of the template. The5'-fixed sequence of oligonucleotides 40N7 and 40N8 also contained a T7RNA polymerase promoter. RNA for the first round of RNA SELEX wastranscribed from double-stranded DNA templates generated by primerextension on single-stranded DNA templates 40N7 and 40N8 with the Klenowfragment of DNA polymerase I. RNA SELEX consisted of up to 15 rounds ofRNA synthesis, binding to target, partitioning of bound and unbound RNAby nitrocellulose filtration, cDNA synthesis, and PCR amplification toregenerate the double-stranded DNA template. Binding to the target bythe RNA pool was performed in binding buffer A (120 mM NaCl,2.5 mMKCl,0.12 mM MgSO4, 40 mM HEPES, 20 mM NaH2PO4/Na2HPO4 pH 7.4, 0.01% HSA)at 37 degrees for at least 10 min prior to filtration. In contrast, thefirst round of single-stranded DNA SELEX was performed by using thesynthetically synthesized oligonucleotides 40D7 and 40D8 directly. SELEXconsisted of 25 rounds of binding to target, partitioning of bound andunbound single-stranded DNA by nitrocellulose filtration, PCRamplification to generate a double-stranded DNA population, andpreparative polyacrylamide gel electrophoresis to purify single-strandedDNA for the next round of SELEX. Binding of the target to thesingle-stranded DNA pool was performed in binding buffer B (150 mMNaCl,10 mM Tris-acetate pH 7.5, 0.001% BSA) at 37 degrees for at least15 rain prior to filtration. Radiolabeling of RNA as well as DNArepertoires was performed by incubation of 5 picomoles nucleic acid, 2units of T4 polynucleotide kinase, and 6 mL γ³² P!ATP (800 Ci/mmol) in avolume of 10 mL at 37 degrees for 30 min. The concentration of nucleicacid at each round of the SELEX experiment varied between 1500 nM and 1nM while the concentration of the target TGFβ1 varied between 150 nM and0.03 nM.

D. Cloning and Sequencing of Ligands

Cloning of the nucleic acid repertoire was performed as described byTuerk and Gold (1990) Science 249:505-510 using double-stranded DNA thatwas generated from the RNA repertoire by PCR amplification.PCR-amplified DNA was digested with the restriction enzymes SphI andHindIII and ligated into compatible sites within pGEM. Ligated plasmidswere transformed into E. coli and plated onto LB agar containing5-bromo-4-chloro-3indolyl β-D-galactoside, isopropyl thiogalactoside,and 100 mg/mL ampicillin. Colonies not expressing β-galactosidase wereanalyzed. Sequencing of DNA was performed as described by Tuerk and Gold(1990) using the dideoxynucleotide procedure of Sanger et al. (1977)Proc. Natl. Acad. Sci. USA 74:5463-5467. Plasmids were isolated from E.coli by the alkaline lysis miniprep procedure (Manitatis et al. (1982)in Molecular Cloning: A laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). DNA was incubated in 50 mM Tris-HCl(pH 8.3), 60 mM NaCl, 6 mM magnesium acetate, and 1 mM DTT with 0.4 mMdNTP and 0.2 mM dideoxy-NTP for 20 min at 48 degrees. DNA polymerase waspresent at 4 units per reaction. The reactions were stopped by theaddition of 10 mL of loading buffer and heated to 95 degrees for 3 minprior to gel electrophoresis on a 6% polyacrylamine/8M urea denaturinggel. G-track sequencing was performed as described and provided aconvenient method to quickly screen the cloned library for ligands ofdifferent sequence. Briefly, the G-track sequencing reaction contained50 mM Tris-HCl (pH 8.3), 60 mM NaCl,6 mM magnesium acetate, and 1 mM DTTwith 0.4 mM dNTP, 0.2 mM dideoxy-GTP, and 4 units of DNA polymerase. Thereaction was performed at 48 degrees for 20 rain and was stopped by theaddition of 10 uL of loading buffer and heated to 95 degrees for 3 minprior to gel electrophoresis on a 6% polyacrylamide/8M urea denaturinggel.

Example 6

BINDING ANALYSIS, BIOASSAY RESULTS, AND SEQUENCES OF A ssDNA LIBRARY

Binding analysis of the 40D7 DNA library for TGF-B1 is shown in FIG. 1.Binding data obtained from round 19 (triangles) and round 0 (circles)are shown. The experiment was performed by incubating nucleic acid (lessthan 1 nM) and the indicated concentration of TGF-β1 in Binding Buffer(150 mM NaCl, 10 mM Tris-acetate pH 8.2, 0.001% BSA) for 15 minutes at37 degrees in a volume of 0.1 mL. Samples were filtered throughnitrecellulose and were immediately followed by 3 mL of TE Buffer (10 mMTris-acetate pH 8.0, 0.1 mM EDTA). The percentage of radiolabel boundwas calculated from the amount of radiolabel retained on thenitrecellulose falter and the total radiolabel added to the bindingreaction. The results show that the apparent Kd of the 40D7 library is 1nM, whereas the starting pool has an apparent Kd of 30 nM. Thus, the40D7 library shows an increase of about three fold in binding.

A PAI-luciferase assay to detect TGFβ-1 activity in the presence of thenucleic acid libraries generated in Example 5 was performed as describedin Abe et al. (1994) Analytical Biochem. 216:276-284. Mink lungepithelial cells containing the PAI-luciferase reporter gene wereincubated with TGF-β1 (10 pM) and oligonucleotides from the DNAlibraries or anti -TGF-B antibody (60 μg/mL). The mink lung epithelialcells were incubated for 18 hours and oligonucleotides werepre-incubated with TGF-β1 before the assay and re-added after 8 hours.Addition of oligonucleotides alone (100 nM) to the cell culture did notaffect the assay (data not shown). The identity of the oligonucleotidelibraries as well as their effect on luciferase activity is indicated inFIG. 2. The ssDNA library 40N7 completely inhibited the activity ofTGF-B1, while the control (an equal concentration of randomized nucleicacid) showed a small stimulation of TGF-B1 activity.

Based on the results of the binding analysis and PAI-luciferase assay,DNA ligands from the 40N7 library were sequenced as described in Example5. The sequences are shown in Table 6 (SEQ ID NO: 55-89). As the DNA40N7 library showed inhibition in the PAI-luciferase bioassay, it isreasonable to suggest that the individual clones from the library areTGFβ1 binders.

                                      TABLE 1                                     __________________________________________________________________________    Nucleic Acid Sequences Used in SELEX Experiments described in Examples        1-4                                                                            ##STR1##                                                                     __________________________________________________________________________     ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                    BamH1 cloning site engineered into pGem9zf to clone SELEX experiments         3-9.                                                                           ##STR11##                                                                    __________________________________________________________________________     *GAUC or GATC, these bases only gauc or gact 62.5% specified base, 12.5%      the other three bases                                                    

                  TABLE 2                                                         ______________________________________                                        RNA SELEX Experiments described in Examples 1-4:                              template, pyrimidine nucleotides, and round cloned.                                              2'substitued                                                                             2'substituted                                                                         Round                                   SELEX exp                                                                             template*  UTP        CTP     cloned                                  ______________________________________                                        lib1    64N6       OH         OH      20                                      lib2    64N6       OH         OH      6                                       lib3    40N6 + 64N6                                                                              F          F       4                                       lib4    40N6 + 64N6                                                                              NH.sub.2   NH.sub.2                                                                              5                                       lib5    64N6       NH.sub.2   NH.sub.2                                                                              13                                      lib6    64N6       F          F       13                                      lib7    64N6       F          NH.sub.2                                                                              14                                      lib8    D-123      OH         OH      6                                       lib9    64N6       NH.sub.2   F       5                                       ______________________________________                                         *Sequences of templates are described in Table 1.                        

                  TABLE 2                                                         ______________________________________                                        RNA SELEX Experiments described in Examples 1-4:                              template, pyrimidine nucleotides, and round cloned.                                              2'substitued                                                                             2'substituted                                                                         Round                                   SELEX exp                                                                             template*  UTP        CTP     cloned                                  ______________________________________                                        lib1    64N6       OH         OH      20                                      lib2    64N6       OH         OH      6                                       lib3    40N6 + 64N6                                                                              F          F       4                                       lib4    40N6 + 64N6                                                                              NH.sub.2   NH.sub.2                                                                              5                                       lib5    64N6       NH.sub.2   NH.sub.2                                                                              13                                      lib6    64N6       F          F       13                                      lib7    64N6       F          NH.sub.2                                                                              14                                      lib8    D-123      OH         OH      6                                       lib9    64N6       NH.sub.2   F       5                                       ______________________________________                                         *Sequences of templates are described in Table 1.                        

                  TABLE 4                                                         ______________________________________                                        Dissociation and Inhibition Constants                                         Group  Ligand      B.sub.max                                                                             K.sub.d IC.sub.50                                  ______________________________________                                        A      lib3-13     0.60    0.9 nM   9.7 nM                                                       0.38    0.7 nM   42 nM                                                        0.55    0.9 nM   18 nM                                                                         32 nM                                            lib3-3      0.44    1.7 nM  NT                                                lib4-32     0.50    0.8 nM   20 nM                                                                        157 nM                                            lib5-5      0.37    2.4 nM   49 nM                                            lib5-7      0.33    3.4 nM   17 nM                                            lib8-9      0.4     1.7 nM  210 nM                                            lib8-9*     0.35    2.8 nM  124 nM                                            lib5-48     0.32    3.8 nM  not inhibitory                                    lib2-6-4    0.20    3.1 nM  not inhibitory                                    lib6-23     0.35    3.4 nM  not inhibitory                                    lib7-21**** 0.18    2.4 nM  not inhibitory                                    lib7-43**** 0.33    3.3 nM  not inhibitory                             B      lib4-12     0.15    0.4 nM  109 nM                                                        0.08    0.2 nM  108 nM                                                                         69 nM                                            lib3-44     0.18    1.3 nM  119 nM                                            lib3-42     0.16    0.6 nM   22 nM                                     C      lib1-20-3** 0.67     30 nM  not inhibitory                                    lib1-20-3-82**                                                                            0.46    6.1 nM  not inhibitory                                    lib6-30**   0.35    8.8 nM  not inhibitory                             D      lib2-6-1*   0.40    14.3 nM 112 nM                                                                        103 nM                                            lib2-6-1-81*                                                                              0.39    10.7 nM 201 nM                                                                        298 nM                                            lib8-23*    0.48    6.6 nM  not inhibitory                                    lib9-10*    0.24    1.1 nM  not inhibitory                             Orphans                                                                              lib3-45     0.08    1.9 nM  not inhibitory                                    lib1-20-5** 0.42     46 nM  not inhibitory                                    lib1-20-12***                                                                             0.34    3.1 nM  NT                                                lib1-6-8*** 0.12    4.7 nM  NT                                         Controls                                                                             lib5-9              nonbinder                                                                             not inhibitory                                    random 64N6         nonbinder                                                                             not inhibitory                             ______________________________________                                         ligands are 2'-NH2 pyrimidines unless otherwise noted                         *2'-NH2UTP, 2'-FCTP,                                                          **2'-F pyrimidines,                                                           ***2'-OH pyrimidines,                                                         ****2'-FUTP, 2'-NH2CTP                                                   

                                      TABLE 5                                     __________________________________________________________________________                                                                SEQ.              DNA oligonucleotides used in Examples 5 and 6.sup.a         ID                Description            Sequence                             NO.               __________________________________________________________________________    40N7 5N7 3N7                                                                     Template for RNA SELEX 5'-primer for PCR 3'-primer for                                        ##STR12##                                43 44 45          40D7 5D7 3D7                                                                     Starting material for DNA SELEX 5'-primer for PCR 3'-primer for                               ##STR13##                                46 47 48          40N8 5N8 3N8                                                                     Template for RNA SELEX 5'-primer for PCR 3'-primer for                                        ##STR14##                                49 50 51          40D8 5D8 3D8                                                                     Starting material for DNA SELEX 5'-primer for PCR 3'-primer for                               ##STR15##                                52 53             __________________________________________________________________________                                                                54                 .sup.a DNA oligonucleotides 40N7 and 40N8 were used to generate the           doublestranded DNA template for in vitro transcription. The 3'-primers 3N     and 3N8 were also used to generate cDNA from the RNA repertoire.              Synthetically synthesized DNA oligonucleotides 40D7 and 40D8 were used        directly as the starting repertoire for the two singlestranded DNA SELEX      experiments. PCR amplification of the selected repertoires used the           appropriate 5'- or 3'-primer. The symbol 40N indicated a 40nucleotide         randomized region within the oligonucleotide.                            

                                      TABLE 6                                     __________________________________________________________________________    TGFβ1 40N7 DNA Selex Sequence of fifty randomly chosen clones.                                                 SEQ. ID                                                                       NO.                                     __________________________________________________________________________    Group A                                                                       20 (11 clones)                                                                        CCAGGGGGGGTATGGGGGTGGTGCTACTTACTTGCGTCTT                                                                    55                                       4      CCAGGGGGGGTATGGGGGTAGTGCTACTTACTTGCGTCTT                                                                    56                                       5      CCAGGGGGGGTATGGGGGTAGTACTACTTACTTACGTCTT                                                                    57                                       8      CCAGGGGGGGTATGGGGGTATACTACTTACTTACGTCTT                                                                     58                                      13      CCAGGGGGGGTATGGGGGTAATACTACTTACTTACATCTT                                                                    59                                      16      CCAGGGGGGGTATGGGGGTAATACTACTTACTTACGTCTT                                                                    60                                      40      CCAGGGGGGGTATGGGGGTGGTGTTACTTACTTGCGTCTT                                                                    61                                      48      CCAGGGGGGGTATGGGGGTGGTGCTTCTTACTTGCGTCTT                                                                    62                                      Group B                                                                       18      CCAGGGGGGGTATGGGGGTGGTGTACTTTTTCCTGCGTCTTC                                                                  63                                      19      CCAGGGGGGGTATGGGGGTGGTTCGTTTTTCTTTGCGGCTT                                                                   64                                      32      CCAGGGGGGGTGTGGGGGTGGTGTACTTTTTCTTGTCTTC                                                                    65                                      46      CCAGGGGGGGTATGGGGGTGGTTTGGTATGTTGCGTCCGT                                                                    66                                      Group C                                                                       12 (3 clones)                                                                         CCGGGGTGGGTATGGGGGTAATACTACTTACTTACGTCTT                                                                    67                                       1      CCGGGGGTGGGTAGGGGGGTAGTGCTACTTACTTACGTCTT                                                                   68                                       3      CCAGGGTCGGTGTGGGGGTAGTACTACTTACTTGCGTCTT                                                                    69                                      10      CCAGGGTGGGTATGGGGGTAGTGCTACTTACTTGCGTCTT                                                                    70                                      23      CCGGGGTGGGTATGGGGGTGGTGCTACTTACTTGCGTCTT                                                                    71                                      34      CCTGGGTGGGTATGGGGGTGGTGCTACTTACTTGCGTCTT                                                                    72                                      Group D                                                                        2      CCACGGGTGGGTGTGGGGTAGTGTGTCTCACTTTACATCAC                                                                   73                                       6      CCCGGGGTGGGTGTGGGGTAGTGTATTATATTTACAGCCT                                                                    74                                      25 & 38 CCAGGGTCGGTGTGGGGTGGTGTACTTTTTCCTGTCCTTC                                                                    75                                       7      CCAGGGTCGGTATGGGGTAGTGTACTTTTTAATGATCTTC                                                                    76                                       9      CCCGGGGGAGAGCGGTGGGTAGTGTTCTATAGTATTCGTGT                                                                   77                                      11      CCAGGGGGGGTATGTTTTTAATACTACTTACTTACGTCTT                                                                    78                                      17      CCAGGGAGGGTATGGGGGTGGTGTTTCTAGTTTTGCGGCGT                                                                   79                                      21      CCAGGGTGGGCATGGGGGTGGTGTGGATTAATTCTTCGTCC                                                                   80                                      24      CCAGGGTCGGTGTGGGGTGGTGTTTTTATTTACTCGTCGC                                                                    81                                      28 & 30 GGGGCGGCTTGGAAGAGGTTGCCGGTTGGAGTATTCGAGC                                                                    82                                      29      CCAGGTGTGGGGTGGTTTGGGTTTTCTTTCGTCGCC                                                                        83                                      31      CCAGGGTGGGTATGGGGGTTTAATTAATTCTTCGTCCCA                                                                     84                                      35      GGGGCGGCTTGGAAGAGGTTGCCGGTTGGAGTATTCGAGC                                                                    85                                      36      CCCGGGGTGGGTGTGGGGTGGTGTGAATTAATTCTTCGTCC                                                                   86                                      41      CCCGGGGTGGGTGTGGGGTGGTGTATTATATTTGCGGCCT                                                                    87                                      44 & 45 CCAGGGTCGGTGTGGGTGGTGTACTTTTTCCTGTCCTTC                                                                     88                                      50      GGGGCGGCTTGGAAGAGGTTGCCGGTTGGAGTATTCGAGC                                                                    89                                      __________________________________________________________________________     Bold typeface indicates a discrepancy with the most common sequence of        that group.                                                              

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 89                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GGGGGAGAACGCGGAUCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGCUUCGCUCUAGAUCU100                         CCCUUUAGUGAGGGUUA117                                                          (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      GGGGGAGAACGCGGAUCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNNNNAAGCUUCGCUCUAGAUCUCCCUUUAGUGAGGGUUA93                                 (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 123 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      GGGGGAGAACGCGGAUCCUGUCUCCACCGCCGAUACUGGGGUUCCUGGGG50                          CCCCUCCAUGGAGGAGGGGGGAGGGGGUGGUUCGGAGAAAGCUUCGCUCU100                         GAAUCUCCCUUUAGUGAGGGUUA123                                                    (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      GGGAGAACGCGGATCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGCTTCGCTCTAGA95                               (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      GGGAGAACGCGGATCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNNAAGCTTCGCTCTAGA71                                                       (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GGGGGAGAACGCGGATCCTGTCTCCACCGCCGATACTGGGGTTCCTGGGG50                          CCCCTCCATGGAGGAGGGGGTGGTTCGGAGAAAGCTTCGCTCTAG95                               (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      TAATACGACTCACTATAGGGGGAGTCTGCGGATCC35                                         (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      TAATACGACTCACTATAGGGGGAGAACGCGGATCC35                                         (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      TAACCCTCACTAAAGGGAGATCTAGAGCGAAGCTT35                                         (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GATTTAGGTGACACTATAGAATATGCATCACTAGTAAGCTTTGCTCTAGA50                          (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     GGATCCCGGAGCTCCCTATAGTGAGTCGTATTA33                                           (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 125 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     GGGGGAGAACGCGGAUCCGAGCAAUCCCAGGCGCAUAGCUUCCGAGUAGA50                          CAGGAGGGAGGGGUGGAUGUGGCGUCUACUCGGUGUCGUGAAGCUUCGCU100                         CUAGAUCUCCCUUUAGUGAGGGUUA125                                                  (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 125 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     GGGGGAGAACGCGGAUCCGAGCAACCCCAGGCGCAUAGCUUCCGAGUAGA50                          CAGGAGGGAGGGGUGGAUGUGGCGUCUACUCGGUGUCGUGAAGCUUCGCU100                         CUAGAUCUCCCUUUAGUGAGGGUUA125                                                  (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 125 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     GGGGGAGAACGCGGAUCCGAGCAACCCCAGGCGCAUAGCUUCCGAGUAGA50                          CAGGCGGGAGGGGUGGAUGUGGCGUCUACUCGGAGUCGUGAAGCUUCGCU100                         CUAGAUCUCCCUUUAGUGAGGGUUA125                                                  (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'-amino                           (2'NH2)modified                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     GGGGGAGAACGCGGAUCCGGCAACCCCAGGCGCAUAGCUUCCGAGUAGAC50                          AGGCGGGAGGGGUGGAUGUGGCGUCACGAGGAAGCUUCGCUCUAGAUCUC100                         CCUUUAGUGAGGGUUA116                                                           (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 123 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     GGGGGAGAACGCGGATCCGCAAUCCCAGGCGCAUAGCUUCCGAGUAGACA50                          GGAGGGAGGGGUGGAUGUGGCGUCUACUCGGCGUCGUGAAGCUUCGCUCU100                         AGAUCUCCCUUUAGUGAGGGUUA123                                                    (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'-amino                           (2'NH2)modified                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     GGGGGAGAACGCGGAUCCGAGCAAUCCCAGGCGCAUAGCUUCCGAGUAGA50                          CAGGAGGGAGGGGUGGAUGUGGCGUCUCGAGGAAGCUUCGCUCUAGAUCU100                         CCCUUUAGUGAGGGUUA117                                                          (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 123 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'-amino                           (2'NH2)modified                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     GGGGGAGAACGCGGAUCCGAGCAAGCCCUGGCAUAGCUUCCGAGUAGACA50                          GGAGGGAGGGGUGGAUGUGGCGUCUACUCGGUGUCGUGAAGCUUCGCUCU100                         AGAUCUCCCUUUAGUGAGGGUUA123                                                    (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 115 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'-amino                           (2'NH2)modified                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     GGGGGAGAACGCGGAUCCGGCAAUCCCAGGCGCAUAGCUUCCGAGUAGAC50                          AGGAGGGAGGGGUGGAUGUGGUGUACGAGGAAGCUUCGCUCUAGAUCUCC100                         CUUUAGUGAGGGUUA115                                                            (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     GGGGGAGAACGCGGAUCCGAGCAAUCCCAGGCGCAUAGCUUCCGAGUAGA50                          CAGGAGGGAGGGGUGGAUGUGGUGUCUCGAGAAGCUUCGCUCUAGAUCUC100                         CCUUUAGUGAGGGUUA116                                                           (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     GGGGGAGAACGCGGAUCCAAGCUUCGAGUAGACAGGAGGGAGGGGUGGAU50                          GUGGAGUCUCGAGAAGCUUCGCUCUAGAUCUCCCUUUAGUGAGGGUUA98                            (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     GGGGGAGAACGCGGAUCCGAGCAAUCCUAAGCAUAGCUUCGAGUAGACAG50                          GAGGGAGGGGUGGAUGUGGCGUCUCGAGAAGCUUCGCUCUAGAUCUCCCU100                         UUAGUGAGGGUUA113                                                              (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-F uracil                                (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'NH2 cytosine                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     GGGGGAGAACGCGGAUCCGAGCAAUCCCGGGCGCAUAGCUUCCGAGGAGA50                          CAGGCGGGAGGGGUGGAUGUGGCGUCUCGAGAAGCUUCGCUCUAGAUCUC100                         CCUUUAGUGAGGGUUA116                                                           (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'-F uracil                                (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'NH2 cytosine                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     GGGGGAGAACGCGGAUCCGAGCAAUCCCAGGCGCAUAGCUUCCGAGUAGA50                          CAGGCGGGAGGGGUGGAUGUGGCGUCUCGAGAAGCUUCGCUCUAGAUCUC100                         CCUUUAGUGAGGGUUA116                                                           (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                     GCUUCCGAGUAGACAGGAGGGAGGGGUGGAUGUGGCGUCUAC42                                  (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                     CUUCCGAGUAGACAGGAGGGAGGGGUGGAUGUGGCGUCUACUC43                                 (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'-amino                           (2'NH2)modified                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                     GGGGGAGAACGCGGAUCCGGCAACCCCAGGCGCAUAGCUUCCGAGUAGAC50                          AGGCGGGAGGGGUGGAUGUGGCGUCACG78                                                (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'-amino                           (2'NH2)modified                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                     GGGGGAGAACGCGGAUCCUGAGAAGGACGUCGGGGUCAACGGGGUGAGGU50                          GCAGCAGAAAGGGCCGGCACCACAUGACGUAAAAGCUUCGCUCUAGAUCU100                         CCCUUUAGUGAGGGUUA117                                                          (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                     GGGGGAGAACGCGGAUCCUGAGAAGGACGUCGGGGUGAGGUGCAGCAGAA50                          AGGGCCGGCACCACAUGACGUAAAAGCUUCGCUCUAGAUCUCCCUUUAGU100                         GAGGGUUA108                                                                   (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                     GGGGGAGAACGCGGAUCCGGUGGGAAAGUCGGAUUAUGUGUGUAGAUUUG50                          UGUGCGAAAGCUUCGCUCUAGAUCUCCCUUUAGUGAGGGUUA92                                  (2) INFORMATION FOR SEQ ID NO: 31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'-amino                           (2'NH2)modified                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                     GGAGAACGCGGAUCCUGAGAAGGACGUCGGGGUCAACGGGGUGAGGUGCA50                          GCAGAAAGGGCCGGCACCA69                                                         (2) INFORMATION FOR SEQ ID NO: 32:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                     GGGGGAGAACGCGGAUCCUGCUAGACCGAGGAUGCAAAGGGACAUGCAUU50                          AGGGAAACCUAUGUAUAAGAACGCGGUCGCAGAAGCUUCGCUCUAGAUCU100                         CCCUUUAGUGAGGGUUA117                                                          (2) INFORMATION FOR SEQ ID NO: 33:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 83 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                     GGGGGAGAACGCGGAUCCUGCUAGACCGAGGAUGCAAAGGGACAUGCAUU50                          AGGGAAACCUAUGUAUAAGAACGCGGUCGCAGA83                                           (2) INFORMATION FOR SEQ ID NO: 34:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                     GGGGGAGAACGCGGAUCCUGCUAGACCGAGGAUGCAAAGGGACAUGCAUU50                          AGGGAAACCUAUUAUAAGAACGCGGUCGCAGAAGCUUCGCUCUAGAUCUC100                         CCUUUAGUGAGGGUUA116                                                           (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-F cytosine                              (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'NH2 uracil                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     GGGGGAGAACGCGGAUCCUGUCUCCACCGCCGAUACUGGGGUUCCUGGGG50                          CCCCUCCAUGCAGGAGGGGGGUGGUUCGGAGAAAGCUUCGCUCUAGAUCU100                         CCCUUUAGUGAGGGUUA117                                                          (2) INFORMATION FOR SEQ ID NO: 36:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-F cytosine                              (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'NH2 uracil                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     GGGGGAGAACGCGGAUCCUGUCUCCACCGCCGAUACUGGGGUUCCUGGGG50                          CCCCUCCAUGCAGGAGGGGGGUGGUUCGGAG81                                             (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-F cytosine                              (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'NH2 uracil                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     GGGGGAGAACGCGGAUCCUGUCUCCACCGCCGAUACUGGGGUUCCUGGGG50                          CCGCUCCAUGCAGGAGGGGGGUGGUUCGGAGAAAGCUUCGCUCUAGAUCU100                         CCCUUUAGUGAGGGUUA117                                                          (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 115 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All C's are 2'-F cytosine                              (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All U's are 2'NH2 uracil                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     GGGGGAGAACGCGGAUCCUGUCUCCACCGCCGAUACUGGGGUUCCUGGGG50                          CCCCUCCAUGCAGGAGGGGUGGUUCGGAGAAAGCUUCGCUCUAGAUCUCC100                         CUUUAGUGAGGGUUA115                                                            (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     GGGGGAGAACGCGGAUCCGGAAGUCUGGUCUUUGGGGAGUCCGCAUGGCC50                          CUGGCGAAAGCUUCGCUCUAGAUCUCCCUUUAGUGAGGGUUA92                                  (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 111 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: All pyrimidines are 2'Fluoro                           (2'-F)modified                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     GGGGGAGAACGCGGAUCCAAGAAUGUUCGGCCGCACGAGGUGACAGUGGU50                          GCGGAUACGGACCGAUUGGGUUUGCCAAGCUUCGCUCUAGAUCUCCCUUU100                         AGUGAGGGUUA111                                                                (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                     GGGGGAGAACGCGGAUCCGGUCACCCGGGCAUAUAACAAUGCCGACACUG50                          GGGUACCUGGGACGGGUGGGACUGGACGGAAGAAGCUUCGCUCUAGAUCU100                         CCCUUUAGUGAGGGUUA117                                                          (2) INFORMATION FOR SEQ ID NO:42 :                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                     GGGGGAGAACGCGGAUCCAUAACCGGCUGCAUGGGAGGGACAUCCUGGGA50                          AAGGACGGGUCGAGAUGACCUGAGCAGUUCCGGCAAGCUUCGCUCUAGAU100                         CUCCCUUUAGUGAGGGUUA119                                                        (2) INFORMATION FOR SEQ ID NO: 43:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                     TCGGGCGAGTCGTCTGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNNCCGCATCGTCCTCCC71                                                       (2) INFORMATION FOR SEQ ID NO: 44:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                     TAATACGACTCACTATAGGGAGGACGATGCGG32                                            (2) INFORMATION FOR SEQ ID NO: 45:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                     TCGGGCGAGTCGTCTG16                                                            (2) INFORMATION FOR SEQ ID NO: 46:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:                                     GGGAGGACGATGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 47:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:                                     GGGAGGACGATGCGG15                                                             (2) INFORMATION FOR SEQ ID NO: 48:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 1-3 is biotin                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:                                     NNNTCGGGCGAGTCGTCTG19                                                         (2) INFORMATION FOR SEQ ID NO: 49:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:                                     GCCTGTTGTGAGCCTCCTGTCGAANNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNNNNNNNNNNTTGAGCGTTTATTCTTGTCTCCC87                                       (2) INFORMATION FOR SEQ ID NO: 50:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:                                     TAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA40                                    (2) INFORMATION FOR SEQ ID NO: 51:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:                                     GCCTGTTGTGAGCCTCCTGTCGAA24                                                    (2) INFORMATION FOR SEQ ID NO: 52:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:                                     GGGAGACAAGAATAAACGCTCAANNNNNNNNNNNNNNNNNNNNNNNNNNN50                          NNNNNNNNNNNNNTTCGACAGGAGGCTCACAACAGGC87                                       (2) INFORMATION FOR SEQ ID NO: 53:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:                                     GGGAGACAAGAATAAACGCTCAA23                                                     (2) INFORMATION FOR SEQ ID NO: 54:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (ix) FEATURE:                                                                 (D) OTHER INFORMATION: N at positions 1-3 is biotin                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:                                     NNNGCCTGTTGTGAGCCTCCTGTCGAA27                                                 (2) INFORMATION FOR SEQ ID NO: 55:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTGGTGCTACTTACTTGC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 56:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTAGTGCTACTTACTTGC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 57:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTAGTACTACTTACTTAC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 58:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTATACTACTTACTTACG50                          TCTTCAGACGACTCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO: 59:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTAATACTACTTACTTAC50                          ATCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 60:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTAATACTACTTACTTAC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 61:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTGGTGTTACTTACTTGC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 62:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTGGTGCTTCTTACTTGC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 63:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTGGTGTACTTTTTCCTG50                          CGTCTTCCAGACGACTCGCCCGA73                                                     (2) INFORMATION FOR SEQ ID NO: 64:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTGGTTCGTTTTTCTTTG50                          CGGCTTCAGACGACTCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO: 65:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:                                     GGGAGGACGATGCGGCCAGGGGGGGTGTGGGGGTGGTGTACTTTTTCTTG50                          TCTTCCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 66:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGGGGGTGGTTTGGTATGTTGCG50                          TCCGTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 67:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:                                     GGGAGGACGATGCGGCCGGGGTGGGTATGGGGGTAATACTACTTACTTAC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO:68 :                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:                                     GGGAGGACGATGCGGCCGGGGGTGGGTAGGGGGGTAGTGCTACTTACTTA50                          CGTCTTCAGACGACTCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO: 69:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:                                     GGGAGGACGATGCGGCCAGGGTCGGTGTGGGGGTAGTACTACTTACTTGC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 70:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:                                     GGGAGGACGATGCGGCCAGGGTGGGTATGGGGGTAGTGCTACTTACTTGC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 71:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:                                     GGGAGGACGATGCGGCCGGGGTGGGTATGGGGGTGGTGCTACTTACTTGC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 72:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:                                     GGGAGGACGATGCGGCCTGGGTGGGTATGGGGGTGGTGCTACTTACTTGC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 73:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:                                     GGGAGGACGATGCGGCCACGGGTGGGTGTGGGGTAGTGTGTCTCACTTTA50                          CATCACCAGACGACTCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO: 74:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:                                     GGGAGGACGATGCGGCCCGGGGTGGGTGTGGGGTAGTGTATTATATTTAC50                          AGCCTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 75:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:                                     GGGAGGACGATGCGGCCAGGGTCGGTGTGGGGTGGTGTACTTTTTCCTGT50                          CCTTCCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 76:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:                                     GGGAGGACGATGCGGCCAGGGTCGGTATGGGGTAGTGTACTTTTTAATGA50                          TCTTCCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 77:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:                                     GGGAGGACGATGCGGCCCGGGGGAGAGCGGTGGGTAGTGTTCTATAGTAT50                          TCGTGTCAGACGACTCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO: 78:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:                                     GGGAGGACGATGCGGCCAGGGGGGGTATGTTTTTAATACTACTTACTTAC50                          GTCTTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 79:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:                                     GGGAGGACGATGCGGCCAGGGAGGGTATGGGGGTGGTGTTTCTAGTTTTG50                          CGGCGTCAGACGACTCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO: 80:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:                                     GGGAGGACGATGCGGCCAGGGTGGGCATGGGGGTGGTGTGGATTAATTCT50                          TCGTCCCAGACGACTCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO: 81:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:                                     GGGAGGACGATGCGGCCAGGGTCGGTGTGGGGTGGTGTTTTTATTTACTC50                          GTCGCCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 82:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:                                     GGGAGGACGATGCGGGGGGCGGCTTGGAAGAGGTTGCCGGTTGGAGTATT50                          CGAGCCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 83:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:                                     GGGAGGACGATGCGGCCAGGTGTGGGGTGGTTTGGGTTTTCTTTCGTCGC50                          CCAGACGACTCGCCCGA67                                                           (2) INFORMATION FOR SEQ ID NO: 84:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:                                     GGGAGGACGATGCGGCCAGGGTGGGTATGGGGGTTTAATTAATTCTTCGT50                          CCCACAGACGACTCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO: 85:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:                                     GGGAGGACGATGCGGGGGGCGGCTTGGAAGAGGTTGCCGGTTGGAGTATT50                          CGAGCCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 86:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:                                     GGGAGGACGATGCGGCCCGGGGTGGGTGTGGGGTGGTGTGAATTAATTCT50                          TCGTCCCAGACGACTCGCCCGA72                                                      (2) INFORMATION FOR SEQ ID NO: 87:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:                                     GGGAGGACGATGCGGCCCGGGGTGGGTGTGGGGTGGTGTATTATATTTGC50                          GGCCTCAGACGACTCGCCCGA71                                                       (2) INFORMATION FOR SEQ ID NO: 88:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:                                     GGGAGGACGATGCGGCCAGGGTCGGTGTGGGTGGTGTACTTTTTCCTGTC50                          CTTCCAGACGACTCGCCCGA70                                                        (2) INFORMATION FOR SEQ ID NO: 89:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:                                     GGGAGGACGATGCGGGGGGCGGCTTGGAAGAGGTTGCCGGTTGGAGTATT50                          CGAGCCAGACGACTCGCCCGA71                                                       __________________________________________________________________________

We claim:
 1. A method of identifying nucleic acid ligands totransforming growth factor beta (TGFβ) comprising:a) preparing acandidate mixture of nucleic acids; b) contacting the candidate mixtureof nucleic acids with TGFβ, wherein nucleic acids having an increasedaffinity to TGFβ relative to the candidate mixture may be partitionedfrom the remainder of the candidate mixture; c) partitioning theincreased affinity nucleic acids from the remainder of the candidatemixture; and d) amplifying the increased affinity nucleic acids to yielda mixture of nucleic acids enriched for nucleic acid sequences withrelatively higher affinity and specificity for binding to TGFβ, wherebynucleic acid ligands of TGFβ may be identified.
 2. The method of claim 1further comprising:e) repeating steps b), c), and d).
 3. The method ofclaim 1 wherein said candidate mixture of nucleic acids is comprised ofsingle stranded nucleic acids.
 4. The method of claim 3 wherein saidsingle stranded nucleic acids are ribonucleic acids.
 5. The method ofclaim 4 wherein said nucleic acids comprise 2'-amino (2'-NH₂) modifiedribonucleic acids.
 6. The method of claim 4 wherein said nucleic acidscomprise 2'-F modified ribonucleic acids.
 7. The method of claim 4wherein said nucleic acids comprise 2'-NH₂ -UTP, 2'-F-CTP modifiedribonucleic acids.
 8. The method of claim 4 wherein said nucleic acidscomprise 2'-F-UTP, 2'-NH₂ -CTP modified ribonucleic acids.
 9. The methodof claim 3 wherein said single stranded nucleic acids aredeoxyribonucleic acids.